Human breast epithelial cell type with stem cell and luminal epithelial cell characteristics

ABSTRACT

Described is a substantially purified human breast epithelial cell (Type I HBEC) displaying the following characteristics: variable cell shape; smooth cell colony boundary; deficiency in gap junctional intercellular communication; positive expression of epithelial membrane antigen and keratin 18; negative expression of keratin 14, α6 integrin and gap junction genes for connexins (Cx26, Cx32 and Cx43); growth promotion by fetal bovine serum; induction by cholera toxin to differentiate into Type II HBEC (prior art); and acquisition anchorage independent growth by Semian virus 40 transfection. Also described is a method of obtaining the above-identified epithelial cells comprising the steps of: a) development of a mixture of human breast epithelial cells from reduction mammoplasty tissues using the MSU-1 medium; b) eliminating stromal fibroblasts by a trypsin (0.002%) and ethylenediamine tetraacetic acid (0.02%) solution; c) separating Type I HBEC from Type II HBEC which attach on culture dishes earlier by collecting Type I HBEC that remain in suspension after trypsinization and prolonged incubation; d) the continuing culture of these cells in MSU-1 medium supplemented with fetal bovine serum, which inhibits the growth of Type II HBEC while promoting the growth of Type I HBEC, gives rise to Type I HBEC. Described also is a new defined medium (the MSU-1 medium) which supports the growth of both Type I and Type II human breast epithelial cells.

LICENSING RIGHTS

The United States government may have licensing rights under thisinvention as provided for in the National Institute of Health ResearchGrants CA 50430 and CA 21104.

This is a division of application Ser. No. 08/308,118 filed Sep. 16,1994 now U.S. Pat. No. 5,650,317.

TECHNICAL FIELD

The invention is directed toward epithelial cells and, in particular,human breast epithelial cells having stem cell characteristics.

BACKGROUND ART

The human breast contains a variety of cell types including luminal andbasal epithelial cells that form the ductal tree. These two types ofepithelial cells are immunocytochemically distinguishable in tissuesections (1) or in enzymatically dissociated single-cell suspensions(2). Antigenic markers that can distinguish these two cell types wouldinclude the epithelial membrane antigen (EMA) and keratin 18 which arepredominantly expressed in luminal epithelial cells (1,2) and keratin 14and α6 integrin which are specifically expressed in basal epithelialcells (1,3). When the expression of these antigenic markers wereexamined in primary human breast carcinomas, it was found that thecarcinoma cells were similar to the luminal epithelial cells in theirexpression of antigens (1,3,4). This evidence can be interpreted asindicating that breast carcinomas are primarily derived from luminalepithelial cells or their precursor cells with similar phenotypes.

Most normal human breast epithelial cell cultures were derived eitherfrom lactational fluids, which contained cells primarily of luminalorigin, or were derived from reduction mammoplasty. Cells from reductionmammoplasty, cultured in the commonly used MCDB 170 (Department ofMolecular, Cellular and Developmental Biology, University of Colorado)(5) or DFCI-1 (Dana-Farber Cancer Institute) (6) media, exhibitpredominantly basal epithelial cell phenotypes (1,4).

Stem cells are undifferentiated cells capable of (a) proliferation, (b)self-maintenance, (c) the production of a large number ofdifferentiated, functional progeny, (d) regenerating the tissue afterinjury, and (e) a flexibility in the use of these options (37).

There is a strong interest in identifying the human hematopoietic stemcell. Having possession of the stem cell will allow for identificationof growth factors associated with its self-regeneration. In addition,there may be as yet undiscovered growth factors associated (1) with theearly steps of dedication of the stem cell to a particular lineage; (2)the prevention of such dedication; and (3) the negative control of stemcell proliferation. The availability of stem cells would be extremelyuseful in bone marrow transplantation, as well as transplantation ofother organs or tissues (e.g., liver regeneration and skin grafting).Stem cells are important and ideal targets for gene therapy, where theinserted genes promote the health of the individual into whom the stemcells are transplanted since the stem cells have longer lifespan. In1992, an Italian research group performed the first human gene transferinvolving stem cells instead of lymphocytes in the hope of curing a 5year old child with the rare genetic disease, adenosine deaminasedeficiency. In addition, the ability to isolate the stem cell may servein the treatment of Fanconi's anemia, Hodgkin's disease, lymphomas andleukemias (e.g., juvenile chronic myelogenous leukemia), as well asother neoplastic conditions (e.g., breast cancer). Thus, there have beenworld-wide efforts toward isolating the human hematopoietic stem cell insubstantially pure or pure form. See U.S. Pat. No. 5,061,620, column 2,lines 3-22.

U.S. Pat. No. 4,411,990 describes a primary assay of human tumor stemcells. Example 1 discloses the treatment of bone marrow cells and theircollection using an appropriate medium which contained fetal calf serumthat have been inactivated by heat. The cells were suspended inadditional horse serum, which contained antibiotics and various aminoacids such as glutamine, asparagine, and the like. Column 10 notes thatthe application of simple in vitro culture technique for studies ofhuman tumor stem cells from primary explants will prove of clinicalimportance.

U.S. Pat. No. 5,061,620 describes human hematopoietic stem cell,assigned to SyStemix, Inc. of California. Stem cells are separated fromdedicated cells and they are maintained by regeneration in a growthmedium. The separation is described in Columns 3 and 4 whereinmonoclonal antibodies are useful for identifying markers associated withparticular cell lineages and/or stages of differentiation. Theantibodies may be attached to a solid support to allow for crudeseparation. Human stem cells are maintained in a medium for culturingthe cells which is described in Column 5, Lines 57 and following whichmedium includes amino acids, vitamins, fetal calf serum and the like.Pluripotent human stem cell regeneration is discussed at Column 6, Lines52 and following.

U.S. Pat. No. 5,081,030 describes release of cells from affinitymatrices. Positive selection of normal marrow stem cells is performedutilizing a monoclonal antibody which selectively recognizes theprogenitor cells.

U.S. Pat. No. 5,087,570 teaches a mammalian stem cell composition whichis isolated with monoclonal antibodies. The antibodies are attached to asolid support to allow for separation.

U.S. Pat. No. 5,185,438 pertains to nucleic acids encoding stem cellreceptor FLK-2. The patent pertains to methods of stimulating theproliferation of primitive mammalian hematopoietic stem cells comprisingcontacting the stem cells with a ligand that binds to a receptor proteintyrosine kinases expressed in primitive mammalian hematopoietic cellsand not expressed in mature hematopoietic cells. A primitivehematopoietic cell is totipotent that is capable of reconstituting allhematopoietic blood cells in vivo. (Column 5, Lines 5-7) Column 8 andfollowing describe the isolation of nucleic acid molecules and proteinsthat encode mammalian stem cell receptors. A source of stem cellsincludes fetal liver, spleen, or thymus cells or adult marrow or braincells. Example 2 in Column 14 indicates a technique for isolating thestem cells from fetal liver. The separated re-suspended tissue istreated with heat-inactivated fetal bovine calf serum to inhibit trypsinactivity. The tissue was resuspended in stromal medium which containsfetal calf serum, human serum, glutamine, sodium pyruvate, non-essentialamino acids and a mixture of antibiotics. The sequences for variousmaterials identified in the patent are shown. Similarly related is U.S.Pat. No. 5,270,458.

U.S. Pat. No. 5,214,133 teaches SCL, a hematopoietic growth anddifferentiation factor. SCL is a new human gene. The SCL gene is ofinterest because it is involved with human stem cell leukemia.

U.S. Pat. No. 5,226,914 describes a method for treating connectivetissue disorders. Culturing and passaging of marrow stromal cells aredescribed in Column 9. The medium that was utilized included fetalbovine serum. A substantial list of medium components is described inColumns 5 and 6.

U.S. Pat. No. 5,258,367 is of interest in that it relates to utilizinguteroferrin and rose proteins for stimulating proliferation ofhematopoietic cells.

U.S. Pat. No. 5,300,422 describes a screening method for controllingagranulocytosis. There the blood is collected and assayed for stem cellactivity to N-desmethylclozapine. The stem cells are identified byspecific markers which are identified with monoclonol antibodies.

Cancer Research 47:1634-1645, 1987, describes the use of x-raylethally-irradiated human fibroblast cell mats to isolate and to cultureputative human fetal kidney epithelial stem cells which are deficient ingap junctional intercellular communication. The medium used was amodified Eagle's minimum essential medium with increased amount of aminoacids and vitamins, and supplemented with sodium pyruvate and fetalbovine serum.

It is an object of the present invention to describe a substantiallypurified human breast epithelial cell type which has stem cellcharacteristics.

It is another object of the present invention to describe a technique toobtain human breast epithelial cells, from reduction mammoplasty, thathave stem cell characteristics by using a serum-free defined medium (theMSU-1 medium as defined below) to grow two types of normal human breastepithelial cells (HBEC) (i.e., Type I cells with stem cell and luminalepithelial cell characteristics and Type II cells with basal epithelialcell characteristics) and by separating these two types of cells intosubstantially pure cultures using two contrasting characters: 1) growthpromotion and growth inhibition of Type I and Type II HBEC,respectively, by fetal bovine serum, and 2) late and early attachment onplastic culture dishes of Type I and Type II HBEC, respectively, aftertrypsinization and subculture.

It is yet another object of the present invention to describe a definedculture medium (the MSU-1 medium) that is comprised of a 1:1 (v/v)mixture of a modified Eagle's MEM and a modified MCDB 153, supplementedwith recombinant human epidermal growth factor, insulin, hydrocortisone,human transferrin, 3,3',5-D.L.-triiodo thyronine and 17-β estradiol. Themedium supports the growth of both Type I and Type II HBEC.

These and other objects will be described hereinafter.

SUMMARY OF THE INVENTION

Described is a substantially purified human breast epithelial cell (TypeI HBEC), derived from reduction mammoplasty, comprised of the followingcharacteristics:

cell morphology: variable in shape;

colony morphology: boundary smooth;

gap junctional intercellular communication: deficient;

fetal bovine serum: growth promotion;

cholera toxin: inducing conversion of Type I HBEC to Type II HBEC; and

when subjected to growth in the MSU-1 medium supplemented with fetalbovine serum, the cell has the following expression of:

epithelial membrane antigen: positive;

keratin 18: positive;

keratin 14: negative;

α6 integrin: negative

gap junction genes (Cx26, Cx32, Cx43): negative.

When transfected by SV40, the Type I HBEC acquired the ability to growin soft agar (anchorage independent growth, AIG⁺) in contrast to Type IIHBEC and those HBEC reported in literature (which are AIG⁻). These SV40transformed Type I HBEC exhibit extended lifespan and a greater tendencyto become immortal and neoplastically transformed. Furthermore, the invitro transformed Type I HBEC and breast carcinoma cell lines (e.g.,MCF-7 and T47D) displayed a phenotype similar to that of Type I HBECrather than those of our Type II HBEC and HBEC commercially available(Clonetics).

This last feature described in the above-paragraph and the ability ofType I HBEC to be induced by cholera toxin to differentiate into Type IIHBEC in addition to their greater proliferation potential indicate thatType I HBEC have stem cell characteristics.

Also described is a method of obtaining the above identified epithelialcell comprising the steps of:

a) development of a mixture of human breast epithelial cells fromreduction mammoplasty tissues using the MSU-1 medium;

b) eliminating stromal fibroblasts by a trypsin (0.002%) andethylenediamine tetraacetic acid (EDTA) (0.02%) solution;

c) separating Type I HBEC (with stem cell and luminal epithelial cellcharacteristics) from Type II HBEC (with basal epithelial cellcharacteristics) by collecting Type I HBEC that remain in suspension anddo not attach on plastic surface for 1-2 days after trypsinization andincubation; and

d) the continued culture of these cells in MSU-1 medium supplementedwith fetal bovine serum gives rise to Type I HBEC with stem cell andluminal epithelial cell characteristics.

Described also is a substantially purified epithelial stem cell havingthe characteristics of the epithelial cell described above.

Also described is a method of obtaining the epithelial stem cellutilizing the steps for obtaining the substantially purified epithelialcell.

Described also is a new defined medium (the MSU-1 medium) that supportsthe growth of two types of normal human breast epithelial cells (Type Iand Type II HBEC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are photograph of the first passage of the cells of thepresent invention (A-right and B) and the prior art (A-left);

FIGS. 2A-2C are photograph of the cells of the present invention (A andC-right) while the prior art (B and C-left) is shown to be derived fromthe present invention;

FIGS. 3A-3B are photograph showing the deficiency of gap junctionalintercellular communication (GJIC) for the cells of the presentinvention (A), while the prior art cells (B) are shown to be competentin GJIC;

FIG. 4 is a photograph of immunofluorescence staining of the cells ofthe present invention (left) as compared with the prior art (right) forthe expression of epithelial membrane antigen (EMA) (top), keratin 18(middle), and keratin 14 (bottom), the positive expression of EMA andkeratin 18 and negative expression of keratin 14 are shown for thepresent invention;

FIGS. 5A-5D are photograph showing immunofluorescence staining of thecells of the present invention (A,B) compared to the prior art (C,D) forthe expression of α6 and β4 integrins, the present invention is negativein α6 integrin expression in contrast to the prior art;

FIGS. 6A-6D show immunofluorescence staining of Simian Virus 40 (SV40)transfected with cells of the present invention (B) and cells of theprior art (D) as contrasted with non-transfected cells of the presentinvention (A) and those of the prior art (no staining shown) (C), forthe expression of SV40 large T-antigen, the SV40 transfected cells arepositive in large T-antigen expression; and

FIGS. 7A-7B are photograph of the cells of the present inventiontransfected by SV40 showing anchorage independent growth on the softagar medium, the prior art did not show anchorage independent growthafter SV40 transfection (data not shown).

DESCRIPTION OF PREFERRED EMBODIMENTS

A culture method to grow two morphologically distinguishable normalhuman breast epithelial cell types derived from reduction mammoplastyhas been developed. Type I cells (present invention) are characterizedby a more variable cell shape, smooth cell colony boundaries, theexpression of epithelial membrane antigen (EMA), keratin 18 and thenon-expression of keratin 14 and α6 integrin, and gap junction genes forCx26, Cx32, and Cx43. In addition, the Type I cells (present invention)were growth stimulated by fetal bovine serum (FBS) and were deficient ingap junctional intercellular communication (GJIC). In contrast, Type IIcells (prior art) were characterized by a uniform cell shape, expressionof keratin 14, α6 integrin and the non-expression of EMA and keratin 18.In addition, Type II cells (prior art) were growth inhibited by FBS andwere proficient in GJIC. Type I cells can be induced by cholera toxin tochange their morphology to a Type II cell morphology. Hence Type I cellsantigenically resemble luminal epithelial cells while the Type II cellsmore closely resemble basal epithelial cells. Type I and Type II cellswere transfected with SV40 DNA. Clones with extended life were obtainedfrom both Type I. and Type II cells by SV40 transfection. Some (2/9) ofthe SV40 transfected Type I cell clones became immortal (>100 cumulativepopulation doubling level), whereas none (0/8) of the SV40 transfectedType II cell clones became immortal. The SV40 transfected Type I andType II cell-derived extended life clones and immortal cell linesphenotypically resembled their parental cells with respect to EMA,keratin 14 and keratin 18 expression and GJIC. Each (9/9) of the SV40transfected Type I cell clones grew in soft agar; none (0/8) of the SV40transfected Type II cell clones were capable of growing in soft agar.These results provide evidence that normal human breast epithelialcells, derived from reduction mammoplasty, can be separated into twomorphologically and antigenically different cell types and that thesetwo different cell types significantly differ in their response to anoncogenic (SV40) stimulus. The ability of Type I HBEC to differentiateinto Type II HBEC, the demonstration of Type I HBEC as target cells forneo-plastic transformation (according to stem cell theory ofcarcinogenesis) in addition to high proliferation potential of thesecells indicate that Type I HBEC (the present invention) have stem cellcharacteristics.

The subject stem cell compositions may find use in a variety of ways.Since the cells are primitive and undifferentiated, they can be used tofully reconstitute an irradiated host and/or a host subject tochemotherapy; or used as a source of cells for specific lineages, byproviding for their maturation, proliferation and differentiation intoone or more selected lineages by employing a variety of factors, such aserythropoietin, colony stimulating factors, e.g., GM-CSF, G-CSF, orM-CSF, interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, etc., orthe like, or stromal cells associated with the stem cells.

The stem cells may also be used in the isolation and evaluation offactors associated with the differentiation and maturation ofhematopoietic cells. Thus, the stem cells may be used in assays todetermine the activity of media, such as conditioned media, evaluatefluids for cell growth activity, involvement with dedication ofparticular lineages, or the like.

The stem cells may be used for the treatment of genetic diseases.Genetic diseases associated with hematopoietic cells may be treated bygenetic modification of autologous or allogeneic stem cells to correctthe genetic defect. For example, diseases such as B-thalas-semia, sickelcell anemia, adenosine deaminase deficiency, recombinase deficiency,recombinase regulatory gene deficiency, etc. may be corrected byintroduction of a wild-type gene into the stem cells, either byhomologous or random recombination. Other indications of gene therapyare introduction of drug resistance genes to enable normal stem cells tohave an advantage and be subject to selective pressure, e.g. themultiple drug resistance gene (MDR). Diseases other than thoseassociated with hematopoietic cells may also be treated, where thedisease is related to the lack of a particular secreted product such asa hormone, enzyme, interferon, factor, or the like. By employing anappropriate regulatory initiation region, inducible production of thedeficient protein may be achieved, so that production of the proteinwill parallel natural production, even though production will be in adifferent cell type from the cell type that normally produces suchprotein. It is also possible to insert a ribozyme, antisense or othermessage to inhibit particular gene products or susceptibility todiseases, particularly hematolympho-tropic diseases.

The epithelial cells of the Type I that are obtained herein are stemcell in nature. Stem cells have utility for skin grafting techniques aswell as for liver regeneration. They have utility for treating thosethat are suffering from anemia, Hodgkin's disease, or juvenile leukemia.

Being able to obtain the putative human breast epithelial stem cells ofthe present invention enables one to further perform research oncarcinogenesis to develop chemopreventive and therapeutic strategies forbreast cancer.

    ______________________________________                                        CULTURE MEDIA                                                                 Type I HBEC (present invention): MSU-1 medium or MSU-1                        with 5% fetal bovine serum;                                                   Type II HBEC (prior art): MSU-1 medium or MSU-1                               with 0.4% bovine pituitary extract and                                        cholera toxin (1 ng/ml)                                                       Components of MSU-1 Medium mg/Liter                                           ______________________________________                                        Ammonium Metavanadate (NH.sub.4 VO.sub.3)                                                                0.000293                                           Calcium Chloride.2H.sub.2 O (CaCl.sub.2.2H.sub.2 O)                                                      143.5                                              Cupric Sulfate.5H.sub.2 O (CuSO.sub.4.5H.sub.2 O)                                                        0.00138                                            Ferrous Sulfate.7H.sub.2 O (FeSO.sub.4.7H.sub.2 O)                                                       0.695                                              Magnesium Chloride.6H.sub.2 O (MgCl.sub.2.6H.sub.2 O)                                                    61                                                 Magnesium Sulfate (anhydrous) (MgSO.sub.4)                                                               48.84                                              Magnesium Sulfate (MnSO.sub.4)                                                                           0.0000755                                          Molybdic Acid.4H.sub.2 O (ammonium)                                                                      0.000618                                            (NH4).sub.6 Mo.sub.7 O.sub.24.4H.sub.2 O!                                    Nickel Chloride.6H.sub.2 O (NiCl.sub.2.6H.sub.2 O)                                                       0.0000594                                          Potassium Chloride (KC1)   127.96                                             Sodium Bicarbonate (NaHCO.sub.3)                                                                         1088                                               Sodium Chloride (NaCl)     7617                                               Sodium Metasilicate.9H.sub.2 O (Na.sub.2 SiO.sub.3.9H.sub.2 O)                                           0.071                                              Sodium Phosphate Dibasic (anhydrous) (Na.sub.2 HPO.sub.4)                                                142.04                                             Sodium Phosphate Monobasic (anhydrous) (NaH.sub.2 PO.sub.4)                                              61                                                 Sodium Selenite (Na.sub.2 SeO.sub.3)                                                                     0.00193                                            Stannous Chloride.2H.sub.2 O (SnCl.sub.2.2H.sub.2 O)                                                     0.0000565                                          Zinc Sulfate.7H.sub.2 O (ZnSO.sub.4.7H.sub.2 O)                                                          0.0719                                             L-Alanine                  13.36                                              L-Arginine.HCl             199.85                                             L-Asparagine.H.sub.2 O     22.5                                               L-Aspartic Acid            15.3                                               L-Cysteine.HCl.H.sub.2 O   21                                                 L-Cystine.2HCl             23.5                                               L-Glutamic Acid            22                                                 L-Glutamine                584.6                                              Glycine                    11.26                                              L-Histidine.HCl.H.sub.2 O  65.04                                              L-Isoleucine               89.19                                              L-Leucine                  71.8                                               L-Lysine.HCl               63.64                                              L-Methionine               20.21                                              L-Phenylalanine            33.92                                              L-Proline                  28.77                                              L-Serine                   42                                                 L-Threonine                42                                                 L-Tryptophan               13.62                                              L-Tyrosine.2Na             49.23                                              L-Valine                   52.06                                              D-Biotin                   0.0073                                             Choline Chloride           7.73                                               Folic Acid                 1.15                                               myo-Inositol               10.51                                              Niacinamide                0.768                                              D-Pantothenic Acid (hemicalcium)                                                                         0.869                                              Pyridoxine.HCl             0.781                                              Riboflavin                 0.094                                              Thiamine.HCl               0.919                                              DL-6,8-Thioctic Acid       0.103                                              Vitamin B-12               0.204                                              Adenine.HCl                15.44                                              Ethanolamine               4.88                                               D-Glucose                  1040                                               HEPES                      3300                                               Phenol Red.Na              0.621                                              Phosphoethanolamine        7.06                                               Putrescine.2HCl            0.081                                              Pyruvic Acid.Na            82.5                                               Sodium Acetate (anhydrous) 150.77                                             Thymidine                  0.364                                              Human Recombinant Epidermal Growth Factor                                                                0.0005                                             Human Transferrin          5                                                  Hydrocortisone             0.5                                                Insulin                    5                                                  17-β estradiol        0.002724                                           3,3',5-triiodo-DL-thyronine                                                                              0.01302                                            ______________________________________                                    

It is to be appreciated that some components of the aforementionedmedium are essential and some components of the medium are not essentialfor cell growth and that the optimum range of amount of each componentmay vary greatly. Therefore, the kind and amount of the components maybe modified without departing from the scope of the invention.

Materials And Methods

Culture Media. The medium used in these studies, identified as "MSU-1"medium, is a 1:1 mixture (v/v) of a modified Eagle's MEM (GIBCO BRL LifeTechnologies, Inc., Grand Island, N.Y.) and a modified MCDB 153 (SigmaChemical Co., St. Louis, Mo.) supplemented with human recombinantepidermal growth factor (0.5 ng/ml) (E-1264, Sigma), insulin (5 ug/ml)(I-1882, Sigma), hydrocortisone (0.5 ug/ml) (H-0888, Sigma), humantransferrin (5 ug/ml) (T-7786, Sigma), 3,3',5-triiodo-D.L.-thyronine(2×10⁻⁸ M) (T-2627, Sigma), and 17 β-estradiol (1×10⁻⁸ M) (E-2257,Sigma). The modified Eagle's MEM (7) contains Earle's balanced saltsolution with 1 mg/ml sodium bicarbonate and 7.64 mg/ml sodium chloride,a 50% increase in all vitamins and essential amino acids (exceptglutamine), and a 100% increase in all nonessential amino acids and 1 mMsodium pyruvate (pH adjusted to 6.5 before the addition of sodiumbicarbonate). The modified MCDB 153 was prepared from commercial MCDB153 (8) powdered medium (M-7403, Sigma), supplemented with 0.1 mMethanolamine (E-6133, Sigma), 0.1 mM phosphoethanolamine (P-0503,Sigma), 1.5×10⁻⁴ M calcium and amino acids, i.e. isoleucine (7.5×10⁻⁴M), histidine (2.4×10⁻⁴ M), methionine (9×10⁻⁵ M), phenylalanine (9×10⁻⁵M), tryptophan (4.5×10⁻⁵ M) and tyrosine (7.5×10⁻⁵ M) (9) (Sigma). ThepH of this medium was also adjusted to 6.5 before the addition of sodiumbicarbonate (1.4×10⁻² M).

Acquisition Processing and Culturing of Human Breast Epithelial Cells(HBEC). All reduction mammoplasty tissues were obtained from femalepatients of 21-29 years of age. A total of 7 reduction mammoplastytissue specimens, from 7 different patients, were examined in thesestudies. The HBEC that were obtained from the 7 reduction mammoplastytissue specimens were designated HME-5, 6, 8, 11, 12, 13 and 14. Thetissue specimens were minced into small pieces with scalpels, thendigested in collagenase-Type IA (C-9891, Sigma) solution (1 g tissue /10 mg of collagenase in 10 ml medium) at 37° C. in a waterbath overnight(16-18 hr). The next morning, the solution containing the digestedtissues was centrifuged to remove the collagenase solution. The cellularpellet was washed once with MSU-1 medium before being suspended in theMSU-1 medium supplemented with 5% fetal bovine serum (FBS) (GIBCO).Subsequently, the cells were plated in two flasks (150 cm²). After a 2hour incubation, the cells (or cell aggregates) which remained insuspension were transferred to 4-6 flasks (75 cm²) for the purpose ofreducing the number of attached fibroblasts. After an overnightincubation, the medium was changed to the FBS-free MSU-1 medium. TheMSU-1 medium was changed once every 2 days for 1 week. Subsequently, thecells were removed with solutions of trypsin (0.01%) (Sigma) andethylenediamine tetraacetic acid (EDTA) (0.02%) (Sigma) and stored insolution phosphate buffered saline (PBS) containing 10% dimethylsulfoxide! in liquid nitrogen. During this week period, virtually all ofthe fibroblasts can be removed by treatment (1-2 times) with dilutedtrypsin (0.002%) and EDTA (0.02%) solution.

To start a culture from stored frozen cells in liquid nitrogen, thecells were thawed and placed in the MSU-1 medium supplemented with 5%FBS for 4 hours for the attachment of residual fibroblasts. Theepithelial cells in suspension were transferred to new flasks andcultured in the FBS-free MSU-1 medium. All cultures were incubated at37° C. in incubators supplied with humidified air and 5% CO₂.

Separation of the Two Types of HBEC. The first passage of HBEC,recovered from liquid nitrogen storage, was plated in MSU-1 mediumsupplemented with 5% FBS. After overnight culture, the cells whichremained in suspension, were transferred to new plates. Continuedculture of these cells in the FBS-containing medium gave rise to onemorphological type of cell. The attached cells, in the overnightculture, cultured in the FBS-free MSU-1 medium supplemented with choleratoxin (1 ng/ml) (Sigma) and 0.4% bovine pituitary extract (Pel-Freez,Rogers, AR) gave rise to a second morphological type of cell. The rarecontaminants of the other cell type in these cultures were removed bymechanically scraping the unwanted small colonies once they weremorphologically recognizable.

Assessment of the Characteristics of Cultured HBEC

1. Morphology. The normal HBEC with the two different types ofmorphology can be observed and easily distinguished under a Nikon phasecontrast microscope without any treatment.

2. Immunocytochemical Analysis. HBEC, grown on 35 mm culture dishes forcolony development or until 50% confluent, were rinsed with PBS andfixed with formaldehyde (3.7% in PBS) for 10 minutes. After treatmentwith 95% ethanol for 5 minutes, the cells were incubated with bovineserum albumin (BSA) (1% in PBS) for 30 minutes. This was followed bytreatment with the primary antibody against the target molecules(prepared in PBS containing 0.1% BSA) for 30 minutes. The cells werethen incubated with biotin-conjugated bridging antibody (Sigma) and thenwith fluorescein isothiocyanate (FITC)-conjugated streptavidin (Sigma)(in PBS with 0.1% BSA) for 30 minutes. All treatments were carried outat room temperature. Following each treatment, the cells were thoroughlyrinsed with PBS (5-6 times). The monoclonal antibodies against keratin14 (C-8791) and keratin 18 (C-8541) were obtained from Sigma. Themonoclonal antibody against EMA was a gift from Dr. M. G. Ormerod of theInstitute of Cancer Research, Royal Cancer Hospital (Sutton, Surrey, UK)(38). The monoclonal antibodies to integrin α6 (BQ16) and β4 (UM-A9)(10) were gifts from Dr. M. Liebert of the University of Michigan, AnnArbor, Mich. Cells for integrin immunostaining were treated differentlythan those for keratin 14, 18 and EMA. These cells were fixed withparaformaldehyde (4% in PBS) for 30 minutes. Subsequently, the cellswere placed in ice-cold methanol for 30 seconds. After permeablization,the cells were washed with PBS and incubated in a 3% BSA and 0.1% Tween20 solution for 1.5 hr to block non-specific binding. Subsequently, thecells were incubated with the primary antibodies against integrin α6 andβ4 for 1.5 hr at room temperature. Without using the bridging antibody,the primary antibody-treated cells were incubated for 1.5 hr in the darkwith FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Lab.,Inc. West Grove, Pa.) (in PBS containing 1% BSA and 0.01% Tween 20). Thefluorescence was detected and analyzed by a ACAS 570 cell analyzer(Meridian Instruments, Okemos, Mich.).

Although antibodies to EMA and α6/β4 integrins were obtained fromprivate sources for this study, they are also commercially available(e.g., EMA: Vector Laboratories, NCL-EMA, Zymed Laboratories, Inc.,18-0017; α6/β4 integrins: Chemicon International, Inc. MAB1973, AB1922).

3. Gap Junctional Intercellular Communication (GJIC). GJIC was studiedby the scrape loading/dy transfer technique previously developed in thislaboratory (11). HBEC were grown to 80% confluency in 35 mm culturedishes, rinsed several times with PBS, and exposed to 0.05% Luciferyellow (Sigma) dye solution in PBS. Several cuts were made on themonolayer of cells with a scalpel in the presence of the dye mixture toload the dye into cells. The cells remained in the dye solution at roomtemperature for four minutes. After removing the dye solution, the cellswere rinsed several times with PBS and examined using Nikon, a phasecontrast epifluorescence microscope to assess the extent of dyetransfer.

Treatment of Cultured HBEC with SV40 DNA

SV40 viral DNA was purchased from GIBCO BRL Life Technologies. Thetransfection was mediated by lipofectin (GIBCO). The two cellular typesof HBEC were plated on 60 or 90 mm plates. When the cells reached 50˜70%confluency (each plate contained approximately 2-3×10⁶ cells), they werewashed with the FBS-free MSU-1 medium twice, then incubated with 3 ml ofFBS-free MSU-1 medium containing SV40 DNA (0.67 ug/ml) and lipofectin(10 ug/ml) at 37° C. for hours. The next day, the lipofectin containingmedium was replaced with the MSU-1 medium (with or without FBS, for thetwo different types of HBEC) and the cells were cultured for 3 days. TheSV40 treated cells were subcultured and replated in 9 cm plates at alower cell density to provide more room for cell growth and colonyformation. The cells which were able to form large colonies could beeasily distinguished from the non-transfected cells which senesced aftersubculture. Developing colonies from the two cellular types of HBECafter SV40 transfection were isolated by the trypsin/glass ring methodfor further characterization. HBEC from three reduction mammoplastyspecimens (from 3 different patients, i.e. HME-11, HME-13, and HME-14)were used for the SV40 transfection. The expression of SV40 largeT-antigen in the SV40 transfected HBEC was examined by immunostainingusing the monoclonal antibody AB-2 (Oncogene Science, Inc., Uniondale,N.Y.). The immunostaining procedure for SV40 large T-antigen is similarto the method used for the keratin 14, 18 and EMA antibodies describedpreviously.

Anchorage Independent Growth (AIG). 0.5% Agarose (Type I, lowelectroendosmosis (EEO) Sigma), prepared in MSU-1 medium at 39° C. wasadded to 60 mm culture dishes and allowed to solidify in the incubator.HBEC cells (2×10⁴), suspended in the medium at 39° C. with 0.33%agarose, were overlaid on top of the hard agar layer. Plates wereincubated at 37° C. and liquid medium was added 3 days after HBECinoculation and renewed every 3 days. After 3˜4 weeks of incubation, theAIG colonies were observed between the two agarose layers and the sizeof the colony was determined.

Assessment of Cell Proliferation Potential. The cumulative populationdoubling level (cpdl) was determined by calculating the initial (Ni) andfinal (Nf) HBEC number using the formula: pdl=1n (Nf/Ni)/1n 2, where inis the natural log. SV40 transfected HBEC, which exhibited higherproliferative activities than non-SV40 transfected HBEC, are referred toas having extended life (EL). SV40 transfected HBEC which werepropagated continuously (a cpdl of 100 or greater) are referred to asbeing normal.

Results

Characteristics of HBEC in Culture

Morphology of the Two Types of HBEC. Single HBEC or HBEC aggregates fromreduction mammoplasty tissues began to proliferate in MSU-1 mediumwithin 2 days. These initial cell cultures were subcultured and storedin liquid nitrogen at day 7. The first passage cells, subcultured fromthe initial culture or thawed from the preserved cells in liquidnitrogen, when cultured in the FBS-free MSU-1 medium, formed twomorphologically distinguishable colonies (FIG. 1A). The first colonytype contained cells that were elongated and variable in shape, lessreflective and less distinctive in cell boundary (FIG. 1A-right and 1B).The cells in this colony are herein referred to as Type I cells. Thesecond colony type, herein referred to as Type II, contained cells thatwere more uniform in cell shape (cobble stone-shaped) and have aconspicuous cell boundary (FIG. 1A-left). The edge of Type I cellcolonies is smooth and appears to be bounded by a layer of elongatedcells in contrast to the non-constrained outline of Type II cellcolonies. The presence of these two types of colonies/cells has beenconsistently observed in all seven HBEC primary cultures examined, i.e.HME-5, 6, 8, 11, 12, 13 and 14. The frequencies of these two types ofcolonies/cells vary among the seven different HBEC cultures, e.g. thefrequencies of Type I/Type II colonies at early passage for HME-5,HME-6, and HME-8 are 45%/55%, 9%/91% and 72%/28%, respectively. Inaddition to Type I and Type II cell colonies, some colonies containingboth Type I and Type II cells were also observed (FIG. 2B and C-left).In these colonies, Type I cells were invariably situated at the centerof the colony and were completely or partially surrounded by Type IIcells. This spontaneously morphological change of Type I cells into TypeII cells has been observed in each of the 7 HBEC cultures.

Development of Enriched Type I HBEC Cultures. One major differentbetween Type I and Type II cells is their growth response to FBS. Type Icells are stimu- lated to grow by FBS whereas Type II cells are growthinhibited by FBS (29). A second difference between Type I and Type IIcells is that after subculture, Type II cells attach to plastic platesearlier than do Type I cells. Many Type I cells still remain insuspension in the medium one day after trypsinization and replating.These cells can be transferred to a new plate to obtain a cultureenriched in Type I cells. Adding FBS to the culture medium (inhibits thegrowth of Type II cells) can further enrich the cultures for Type Icells.

Induction By Cholera Toxin of Type I HBEC Into Type II HBEC. The firstpassaged HBEC (HME-5 and 8) were inoculated in MSU-1 medium in thepresence or absence of cholera toxin (1 ng/ml). After 10-12 days, thefrequencies of three types of colonies (Type I cells only, Type II cellsonly and both Type I and Type II cells) were determined. The resultsfrom these experiments consistently showed that cholera toxinsignificantly (P<0.01) increased the frequency of colonies containingboth types of cells (Table 1). The frequency of colonies exemplified byType I cells surrounded by Type II cells, in the presence of choleratoxin, was increased from 28% to 43%, 11% to 16% and 3% to 9%,respectively. The total number of colonies was not significantlyinfluenced by cholera toxin. In the second experiment, HBEC (HME-14)enriched for Type I cells were inoculated in MSU-1 medium in thepresence or absence of cholera toxin (1 ng/ml). After 10 days, in thepresence of cholera toxin, the frequency of colonies with Type II cellswas found to be significantly (P<0.01) increased (from 10% to 18%),while the total number of colonies was not significantly changed (Table1). These results provide evidence that treatment with cholera toxinenhances the transition of Type I cells into Type II cells.

                                      TABLE 1                                     __________________________________________________________________________    Effect Of Cholera Toxin On Frequencies of Type I, Type II and                 Type 1/Type II Colonies Developed From Early Passage HBEC                                               Colony Type -                                                                 Type I Cells                                              Cholera Toxin                                                                        Total Colonies                                                                       Type I                                                                              Surrounded By                                                                        Type II                                      HBEC  (1 ng/ml)                                                                            (No. of Plate)                                                                       Cells Only                                                                          Type II Cells                                                                        Cells Only                                   __________________________________________________________________________    HME-5.sup.a                                                                         -      301 (4)                                                                               95 (31%)                                                                            83 (28%)                                                                            123 (41%)                                          +      362 (4)                                                                               44 (12%)                                                                           155 (43%)                                                                            163 (45%)                                    HME-8.sup.a                                                                         -      1477 (4)                                                                             940 (64%)                                                                           161 (11%)                                                                            376 (25%)                                          +      1941 (4)                                                                             1147 (59%)                                                                          315 (16%)                                                                            479 (25%)                                    HME-8.sup.a  1918 (10)                                                                            1421 (74%)                                                                          54 (3%)                                                                              443 (23%)                                                 1716 (10)                                                                            1249 (73%)                                                                          150 (9%)                                                                             317 (18%)                                    HME-14.sup.b                                                                        -      723 (3)                                                                              652 (90%)     71 (10%)                                          +      666 (3)                                                                              547 (82%)    .sup. 119 (18%).sup.c                        __________________________________________________________________________     .sup.a Early passage HBEC (HME5 and 8) were inoculated in MSU1 medium         supplemented with 5% FBS for 1 day. The medium was changed to MSU1 with o     without cholera toxin the next day, and the cells were incubated for a        total of 10 days for colony development. The colonyforming efficiencies       were 15% and 18% in the absence and presence of cholera toxin,                respectively for HME5.                                                        .sup.b HBEC (HME14) enriched for Type I cells were inoculated in MSU1         medium supplemented with 5% FBS for 1 day. The medium was changed to MSU1     with or without cholera toxin the next day, and the cells were incubated      for a total of 10 days for colony development.                                .sup.c The frequencies of Type 1/Type II cell colonies (HME5 and 8) or        Type II cell colonies (HME14) are significantly higher upon cholera toxin     treatment (P < 0.01) (12).                                               

GJIC in Type I and Type II HBEC. Type I cells and Type II cells wereexamined for their ability to perform GJIC using the scrape loading/dyetransfer technique. The results show that Type I cells were deficient inGJIC (FIG. 3A), while Type Ii cells were efficient in GJIC (FIG. 3B).This difference in GJIC in Type I and Type II cells was observed in eachof the 7 HBEC (HME-5, 6, 8, 11, 12, 13 and 14) and was observed in bothearly and late passaged cells.

Expression of EMA, Keratins 14 and 18 and αb/β4 Integrins in Type I andType II HBEC. Three HBEC (HME-5, 11 and 12) were examined for theirantigenic marker expression. The results showed that Type I cellsconsistently expressed EMA and keratin 18 (luminal epithelial cellmarkers) but not keratin 14 (basal epithelial cell marker), while TypeII cells consistently expressed keratin 14 but not EMA and keratin 18(FIG. 4). Type I cells did not express α6 (basal epithelial cell marker)or β4 integrins (FIG. 5A and B) whereas Type II cells consistentlyexpressed α6 integrins (FIG. 5C) but did not express β4 integrins (FIG.5D). That Type I and Type II cells are distinctly different in theirantigenic expression has been demonstrated in these studies. A summaryof the differences between Type I and Type II cells is provided in TableII.

                  TABLE II                                                        ______________________________________                                        Summary of Differences Between Type I and Type II HBEC                                   Type I     Type II                                                 ______________________________________                                        Cell morphology                                                                            Variable in shape                                                                          Uniform in shape,                                                             cobble-stone appearance                             Colony morphology                                                                          Boundary smooth                                                                            Boundary not smooth                                 Attachment on plastic                                                                      Late         Early                                               surface after                                                                 trypsinization                                                                Effect of fetal bovine                                                                     Growth promotion                                                                           Growth inhibition                                   serum                                                                         Effect of cholera toxin                                                                    Induces Type I cells to change                                                into Type II cell morphology                                     Gap junctional inter-                                                                      Deficient    Efficient                                           cellular communication                                                        Expression of:                                                                Epithelial Membrane                                                                        +            -                                                   Antigen                                                                       Keratin 18   +            -                                                   Keratin 14   -            +                                                   α6 Integrin                                                                          -            +                                                   ______________________________________                                    

Effect of Transfection of Type I and Type II HBEC with SV40 DNA

Isolation of SV40 Transfected HBEC Clones. Type I and Type II cells,from 3 HBEC (HME-11, 13 and 14) were transfected with SV40 DNA. AfterSV40 transfection, the majority of the cells became senescent andstopped proliferating within a week. Against the background of senescentcells, a few colonies containing proliferating cells became evident.These colonies (clones) were isolated for continual growth expansion andfurther characterization. A total of 9 independent Type I cell-derivedSV40 transfected colonies were isolated from HME-11 (referred to asM11SV-1, 2, 3, 4 and 5), HME-13 (referred to as M13SV-1 and 2) andHME-14 (referred to as M14SV-1 and 2). A total of eight independent TypeII cell-derived SV40 transfected colonies were isolated from HME-11(referred to as M11SV-21 AND 22), HME-13 (referred to as M13SV-21, 22and 23) and HME-14 (referred to as M14SV-21, 22 and 23).Immunocytochemical analysis, using antibodies against SV40 largeT-antigen, showed that each of the isolated clones were positive for theexpression of SV40 large T-antigen whereas the non-transfected parentalcells were negative (FIG. 6, Table III). The expression of SV40 largeT-antigen in the SV40 transfected Type I and Type II cells wererestricted to the nuclei.

                  TABLE III                                                       ______________________________________                                        Summary of Characteristics of SV40                                            Transfected Type I and Type II HBEC                                           SV-40                                                                         T-antigen     EMA    K-18      K-14 GJIC  AIG                                 ______________________________________                                        TYPE I                                                                        M11SV-1 +         +      ++      -    -     +                                 M11SV-2 +         +      ++      -    -     +                                 M11SV-3 +         +      ++      -    -     +                                 M11SV-4 +         +      ++      -    -     +                                 M11SV-5 +         +      ++      -    -     +                                 M13SV-1 +         +      ++      -    -     +                                 M13SV-2 +         +      ++      -    -     +                                 M14SV-1 +         +      ++      -    -     +                                 M14SV-2 +         +      ++      -    -     +                                 TYPE II                                                                       M11SV-21                                                                              +         -        .sup. -(+)                                                                          +    N.D.  -                                 M11SV-22                                                                              +         -        .sup. -(+)                                                                          +    +     -                                 M13SV-21                                                                              +         -      -       +    N.D.  -                                 M13SV-22                                                                              +         -      -       +    +     -                                 M13SV-23                                                                              +         -      -       +    +     -                                 M14SV-21                                                                              +         -      -       +    N.D.  -                                 M14SV-22                                                                              +         -      -       +    N.D.  -                                 M14SV-23                                                                              +         -      -       +    N.D.  -                                 ______________________________________                                         *abbreviations:                                                               EMA, epithelial membrane antigen;                                             K18, keratin 18;                                                              K14, keratin 14;                                                              GJIC, gap junctional intercellular communication;                             AIG, anchorage independent growth;                                            N.D., not done;                                                               +, positive,                                                                  ++, strong positive;                                                          -, negative;                                                                  -(+), heterogeneous.                                                     

Proliferation Characteristics of SV40 Transfected Type I and Type IIHBEC. The cpdl was determined for each of the clones of SV40 transfectedType I and Type II cells. Almost all of the SV40 transfected clones hadan extended lifespan, a phenomenon independent as to whether or not theclones were derived from Type I or Type II cells, maximum cpdl rangingfrom 20˜50, i.e., Type I, cpdl, mean ±S.E.=34.4±2.7; Type II, cpdl, mean±S.E.=27.2±2.1). Two of the clones (2/9) (M13SV-1 and M13SV-2), bothderived from SV40 transfected Type I cells, became immortal (with andwithout crisis for M13SV-1 and M13SV-2, respectively), having a cpdlgreater than 100. None of the Type II cell-derived SV40 transfectedclones (0/8) became immortal.

Characterization of SV40 Transfected Type I and Type II Cells. Theexpression of EMA, keratin 18, keratin 14 and the ability to performGJIC in the SV40 transfected Type I cell clones and Type II cell cloneswere found to mimic their parental counterparts, i.e., the expression ofEMA, keratin 18 and GJIC deficiency was observed in Type I cell clonesand the expression of keratin 14 and GJIC proficiency was observed inthe Type II cell clones. These data are summarized in Table III.

Anchorage Independent Growth (AIG) of SV40 Transfected Type I and TypeII HBEC. SV40 transfected Type I and Type II cells had markedlydifferent abilities to grow in soft agar (AIG). Each of the SV40transfected Type I cells formed colonies in soft agar (FIG. 7, TableIII). The colony-forming frequency was, mean ±S.E., 7.2±2.8%. Thelargest colonies observed were ranged from 0.5 mm to 1 mm in diameter.In contrast, none of the SV40 transfected Type II cells formed coloniesin soft agar.

Discussion

As summarized in Table 2, Type I and Type II HBEC are quite differentphenotypically. Type I cells express EMA and keratin 18 but do notexpress keratin 14 and α6 integrin. EMA and keratin 18 expression aremarkers for luminal epithelial cells (1,2). Type II cells expresskeratin 14 and α6 integrin but do not express EMA and keratin 18.Keratin 14 and α6 integrin expression are markers for basal epithelialcells (1,3). In previously published reports in which cultures ofprimary HEEC were examined, it appears that the vast majority of thecultured cells, derived from reduction mammoplasty, more closelyresembled, phenotypically, Type II cells as judged by cell morphology,antigenic expression, and growth inhibition by FBS (1, 4-6).

Cultures of primary HBEC, in which the predominant phenotype is similarto Type I cells, heretofore has not been reported. Without being boundto any theory, it is believed that the reason for this may be due to thetype of culture media used. The media most often used to culture primaryHBEC (e.g., MCDB 170 (molecular, cellular and developmental biology),DFCI-1 (Dana-Farber Cancer Institute) (5,6) are most often supplementedwith cholera toxin and/or bovine pituitary extract. These supplementsfavored the conversion of Type I to Type II cells. Indeed, an importantobservation in our studies is that one can accelerate the spontaneouschange of Type I cells into Type II cells by the addition of choleratoxin to the culture medium. This not only results in a change incellular morphology, but, in addition, results in a striking change ingene expression, e.g., the switch from EMA and keratin 18 expression tokeratin 14 and α6 integrin expression. Furthermore, these cells becomealtered in their responsiveness to serum, i.e. Type I cells are growthstimulated by FBS while the growth of Type II cell is inhibited by thisserum supplement. This conversion may be triggered by an increase in thecellular levels of cyclic AMP induced by cholera toxin. The culturemedium disclosed herein (MSU-1) allows for the separation and/orenrichment of Type I and Type II cells upon supplementing the mediumwith FBS or cholera toxin/bovine pituitary extract.

The induction of immortalization of normal HBEC has been reportedpreviously (13-22). In these studies, either HBEC derived fromlactational samples (milk cells) or HBEC derived from reductionmammoplasty were utilized. Cultured milk cells most often expresskeratin 18 (luminal epithelial cells); on occasion, colonies of culturedmilk cells will also express keratin 14 (1). Cells from reductionmammoplasty contain predominantly basal epithelial cells when culturedin MM or MCDE 170 medium (1). Utilizing the MSU-1 culture medium with orwithout FBS, one can enrich for these two types of cells (luminal andbasal epithelial cells) and determine their differential response to apotential oncogenic stimulus.

SV40 is an oncogenic agent that has been reported by a number oflaboratories to induce immortalization of primary HBEC (13, 16-22).After transfection of SV40 DNA into Type I and Type II cells, cloneswith extended lifespan were derived from Type I and Type II cells atcomparable frequency. However, 2 of 9 of the SV40 transfected Type Icell-derived extended life clones converted to immortal cell lines,while none (0/8) of the SV40 transfected Type II cell-derived extendedlife clones became immortalized. Significantly, however, is thedifference in anchorage independent growth (AIG) between SV40transfected Type I and Type II cells. None (0/8) of the SV40 transfectedType II cell-derived extended life clones displayed AIG, while each(9/9) of the SV40 transfected Type I cell-derived extended life clonesshowed AIG. AIG is often used to identify tumor cells (23) and isfrequently described as a marker for neoplastic transformation (24-26).In past reports in which SV40 was effective in the immortalization ofHBEC, these immortalized cells were incapable of AIG (16-22), with thepossible exception of one cell line which was selected in soft agar(13). Thus, these results demonstrate that Type I cells and Type IIcells differ substantially in their response to an oncogenic stimulus(SV40), particularly with regard to AIG.

The SV40 transfected Type I and Type II cells closely resembled thephenotypes of their parental cells, such as the expression of EMA andkeratins. In addition, they maintained the parental phenotype withregard to GJIC. The Type I cell-derived SV40 transfected clones also aresimilar in many respects to human breast carcinoma cell lines. Forexample, MCF-7 (Michigan Cancer Foundation, Detroit, Mich., AmericanType Culture Collection, HTB 22) and T47D (American Type CultureCollection, HTB 133) human breast carcinoma cell lines, which expressthe antigenic markers keratin 18 and EMA but not keratin 4 are deficientin GJIC and have AIG. While applicant does not wish to be bound to anytheory, these observations suggest that the origin of human breastcarcinomas is the luminal epithelial cell or its precursor cell and thatthe Type I cells described might be the major target cell for neoplastictransformation.

It should also be noted that SV40 transfected Type I cell clones(extended life or immortal) were shown to be non-tumorigenic wheninoculated into athymic nude mice. However, one Type I cell-derived SV40transfected extended life clone (M11SV-1) was treated with a combinationof BrdU and black light, a potent mutagenic treatment (27). An immortalcell line has been obtained from this treatment and this cell line hasbeen shown to be weakly tumorigenic in athymic nude mice. Infection ofthe BrdU/black light immortalized cell line with a mutated ratneuoncogene (28) greatly enhanced the tumorigenicity of these cells uponinoculation into athymic nude mice (29).

Cancer cells are believed to arise from stem cells or early precursorcells and often have a phenotype similar to normal undifferentiatedcells (30) or have a combined phenotype of different cell types of acommon lineage (e.g., leukemia cells often express both lymphoid andmyeloid cell antigens) (31). Therefore, cancer has been termed a diseaseof the pluripotent stem cell (31), a disease of cell differentiation(32) or oncogeny as blocked or partially blocked ontogeny (33). Forhuman tissues, except for peripheral blood stem cells (34), stem cellsof solid tissues have rarely been characterized. Attempts tocharacterize subpopulations of cells with stem cell characteristics insolid tissues have been reported for fetal kidney epithelial cells (35)and for epdiermal cells (36). The phenotype of Type I cells herein issuggestive of the presence of stem cells in the Type I cell populationas some of them have the ability to give rise to the other type of cellswith a different phenotype, i.e. Type I cell (expresses luminalepithelial cell markers) to Type II cell (expresses basal epithelialcell markers). In addition, Type I cells are efficient in GJIC. GJICdeficiency has been reported to be a characteristic of putative stemcells (35).

The results show that two types of HBEC were derived from and culturedfrom human breast reduction mammoplasty. Type I cells have antigeniccharacteristics of luminal epithelial cells while Type II cells haveantigenic characteristics of basal epithelial cells. Importantly, thesetwo types of cells differ substantially in their response to anoncogenic (SV40) stimulus, i,e. the Type I cells have a greater tendencyto become immortal, and most strikingly, have the ability to grow insoft agar (AIG); SV40 transfected Type II cells totally lack the abilityto grow in soft agar (AIG⁺). The ability to separate HBEC into celltypes that vary in their sensitivity to oncogenic stimuli willfacilitate the ability to consistently and reproducibly transform normalHBEC by oncogenic agents.

Details as to the Figures are as follows:

FIG. 1: Representative first passage of HBEC in MSU-1 medium for 5 days(X˜90). Both Type I (A right, and B) and Type II (A-left) coloniesdeveloped in this medium.

FIG. 2: HBEC colonies grown in MSU-1 medium for 8 days (X˜90). One typeof colony contains only Type I cells (A and C-right), the other coloniescontain both Type I and Type II cells (B and C-left). In these lattercolonies, the Type I cells were either partially or completelysurrounded by Type II cells.

FIG. 3: Representative gap junctional intercellular communication (GJIC)in Type I and Type II HBEC as examined by the scrape loading/dyetransfer technique. Type I cells were deficient in GJIC (A). Type IIcells were efficient in GJIC (B) (X˜90).

FIG. 4: Representative expression of EMA (top), keratin 18 (middle) andkeratin 14 (bottom) in Type I (left) and Type II (right) HBEC asrevealed by immunofluorescence staining and detected by the ACAS-570laser cytometer. (X˜200).

FIG. 5: Representative immunofluorescence staining of Type I (A,B) andType II (C,D) HBEC using antibodies against α6 integrin (A,C) and β4integrin (B,D). (X˜200).

FIG. 6: Representative immunofluorescent staining of SV40 transfectedType I HBEC (B) and SV40 transfected Type II HBEC (D). Non-transfectedparental Type I HBEC (A) and Type II HBEC (C) did not show staining.(X˜200).

FIG. 7: Representative anchorage independent growth (AIG) of SV40transfected Type I HBEC (A, X˜36) (B, X˜90).

While the forms of the invention herein disclosed constitute presentlypreferred embodiments, many others are possible. It is not intendedherein to mention all of the possible equivalent forms or ramificationsof the invention. It is understood that the terms used herein are merelydescriptive rather than limiting, and that various changes may be madewithout departing from the spirit or scope of the invention.

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What is claimed is:
 1. A substantially purified human breast epithelialcell comprised of the following characteristics:cell morphology:variable in shape; colony morphology: boundary smooth; and whensubjected to growth in a medium comprised of fetal bovine serum, thecell has the following expression of:epithelial membrane antigen:positive; keratin 18: positive; and keratin 14: negative.
 2. Asubstantially purified human breast epithelial stem cell comprised ofthe following characteristics:cell morphology: variable in shape; colonymorphology: boundary smooth; and when subjected to growth in a mediumcomprised of fetal bovine serum, the cell has the following expressionof:epithelial membrane antigen: positive; keratin 18: positive; andkeratin 14: negative.
 3. The cell of claims 1 or 2 further characterizedas having a delayed adhesion to plastic after trypsinization andsubculture.
 4. The epithelial cell of claim 1 is further characterizedas having a positive response to immunoclonal antibodies against keratin18 and not having a positive response to immunoclonal antibodies againstkeratin
 14. 5. The epithelial cell of claim 2 being furthercharacterized as being converted from a cell morphology variable inshape to a cell morphology uniform in shape and a colony morphology ofsmooth boundary to a colony morphology of boundary not smooth bysubjecting the cell to cholera toxin.
 6. A method of obtaining the cellof claim 1 comprising the steps of:a) providing a mixture of cells; b)subjecting the cells to a culture medium comprised of a mixture ofEagle's MEM, epidermal growth factor, insulin, hydrocortisone,transferrin, triiodothyronine, estradiol, vitamins, and amino acids; c)separating the cells; and d) recovering the desired cells.
 7. A methodof obtaining the epithelial stem cell of claim 2 comprising the stepsof:a) providing a mixture of cells; b) subjecting the cells to a culturemedium comprised of a mixture of Eagle's MEM, MCDB 153, epidermal growthfactor, insulin, hydrocortisone, transferrin, triiodothyronine,estradiol, vitamins, and amino acids; c) separating the cells; and d)recovering the desired cells.
 8. The method of claims 6 or 7 wherein thecells are subjected to trypsinization and subculture in the presence ofa plastic substrate whereby the desired epithelial cell remains in theliquid phase.
 9. The method of claim 8 wherein the cell desired to beseparated is suspended in the liquid phase and is obtained from theliquid phase.
 10. The method of claims 6 or 7 wherein after the cellsare subjected to the culture medium,promoting growth of the cell byadding an effective growth promoting amount of fetal bovine serum, andalso inhibiting the growth of undesired epithelial cell types.
 11. Aculture medium for culturing a human breast epithelial cell comprised ofa mixture of Eagle's MEM, epidermal growth factor, insulin,hydrocortisone, transferrin, triiodothyronine, estradiol, vitamins, andamino acids.
 12. A medium comprising a mixture of Eagles'MEM and MCDB153 medium supplemented with human recombinant epidermal growth factor,insulin, hydrocortisone, human transferrin, triiodothyronine and 17-βestradiol in the absence of prostaglandin E₁, ovine prolactin, bovinepituitary extract and cholera toxin.